PhD Thesis
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Item RNA mediated resistance against banana bunchy top virus (BBTV) in banana(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2024-02-17) Aparna, K Gokul; Vimi LouisThe study entitled “RNA mediated resistance against Banana bunchy top virus (BBTV) in banana” was conducted at the Department of Plant Pathology, College of Agriculture, Vellanikkara and National Research Centre for Banana, Trichy during the period from 2018 to 2023. The study aimed at developing banana lines that have resistance against Banana bunchy top virus (BBTV) through RNAi, using the ligation independent cloning (LIC) method. The RNAi approach targeted the replicase gene of BBTV. The process began with the amplification of replicase gene fragment that contains the dicer substrate siRNA region. Gene specific primers with adaptor sequences were used to amplify the DNA fragment, creating the sense and antisense fragments of the RNAi construct. The LIC vector which specifically contained four adaptor sequences (LIC1 to LIC4) was linearized with SmaI followed by treatment with T4 DNA polymerase and dTTP. Simultaneously, the amplified gene fragments were treated with T4 DNA polymerase in the presence of dATP, facilitating the development of 5' extending single stranded tails. Subsequently, the T4 DNA polymerase treated vector and gene fragments with sticky ends were mixed and incubated. The splicing of the cohesive ends on the insert fragments and the vector resulted in the circularization of the vector. The prepared vector, housing the desired sense and antisense fragments was transformed into E. coli DH5α. All the transformed colonies, obtained on antibiotic selection medium underwent initial screening via colony PCR, demonstrating cent per cent transformation. Further confirmation involved plasmid isolation, verification of intron, restriction digestion and sequencing. The next phase was transforming Agrobacterium EHA105 strain with the prepared vector by modified freeze-thaw method. The success of transformation was confirmed by verifying the presence of sense and antisense fragments through colony PCR. For plant transformation, embryogenic calli of banana cv. Nendran were developed from the immature male inflorescence of 0.5 cm in size, by culturing on MS medium supplemented with different combinations of hormones viz., 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA) and picloram. The embryogenic calli development was observed after five months, with the highest per cent in MS medium with 2,4-D (2 mg l-1) and IAA (1 mg l-1). In order to minimize the time delay, the embryogenic cell suspension (ECS) obtained from NRCB, Trichy was used for plant transformation. The co-cultivation of Agrobacterium carrying the desired construct and ECS was done in the presence of acetosyringone for three days, in the dark at 24 to 25 ⁰C, followed by washing of excess Agrobacterium with cefotaxime. The co-cultivated embryogenic cells were transferred to a selection medium containing kanamycin (100 mg l-1). The transformed selected embryogenic cells were then transferred to the embryogenesis medium for 35 to 40 days, followed by culturing on embryo germination medium, under light. The germinated embryos were cultured in Petri plates for one and half months, followed by regular subculturing and maintenance in tissue culture bottles, till they attained the three to four leaf stage. The plants were rooted in a rooting medium for 30 to 40 days, and then acclimatized in a containment facility. The PCR assay with the primers for amplification of sense and antisense fragments confirmed the presence of RNAi construct in the acclimatized plants. These plants were then tested for BBTV resistance by aphid (Pentalonia nigronervosa) challenge inoculation. The transformed plants remained healthy, while the typical bunchy top symptoms appeared within 25 days on the non-transgenic control plants. However, it was observed that two out of the forty eight transgenic lines gradually developed yellowing symptoms, indicating varying levels of resistance among the transformation events. Therefore, further evaluation of these transgenic lines is crucial to better understand the resistance levels. The study has successfully demonstrated the efficacy of transgenic lines in conferring BBTV resistance through RNAi approach. By utilizing the ligation independent cloning, the hairpin construct preparation was done in more efficient manner. The transgenic lines developed in the study represent a promising tool, offering the scientific community a proactive defense against unmanageable BBTV epidemics in the absence of resistant lines.Item Evaluation of phylloplane microorganisms for the management of early blight of Tomato (Solanum lycopersicum L.)(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2024-08-14) Wongamthing, R.; Sainamole Kurian, PTomato (Solanum lycopersicum L.) is a widely grown vegetable and the most remunerative solanaceous crop in both tropics and sub tropics of the world. It is vulnerable to various biotic diseases among which, early blight caused by Alternaria solani is a destructive one. Management of diseases using biocontrol agents is a safe alternative in the integrated disease management system. However, success of a bioagent is dependent on its comparative survivability in the new niche. Therefore, the present investigation was undertaken to identify strains of beneficial microorganisms adept on the phylloplane of tomato for use against early blight as it is a disease affecting mainly the foliage. As part of the investigation, purposive sampling survey was conducted in Thrissur and Palakkad districts of Kerala. It was found that early blight is prevalent in all the areas surveyed and the disease severity on foliage varied from 23 to 78 per cent at various locations. Though several fungi were found to be associated with the disease, only six isolates collected from Pudukkad, Valapad, Mundathikode, Vadakarapathy, Nenmara and Parrisakal were identified as Alternaria sp.(PDK), A. alternata (VAL), Fusarium oxysporum (MUN), A. solani (VAD and NEM) and Corynespora cassiicola (PAR) which could be proved as pathogens on tomato. Phylloplane microorganisms were isolated from both the healthy and diseased leaves collected from plants at various locations and a total of 132 isolates were then subjected to preliminary in vitro screening for their ability to suppress A. solani and 59 were selected as they could antagonize the pathogen in various degrees. The antagonists thus selected were further screened by dual culture technique and 36 showed more than 60 per cent inhibition. The antagonism exerted by phylloplane isolates was clearly discerned by limited growth, or the complete absence of fungal mycelium in the inhibition zone between the pathogen and the test isolate. Out of the 36, five were selected as promising potential antagonists as they could give more than 80 per cent inhibition of the pathogen. The selected five are identified as Trichoderma spp. (PF2 and PRF1), Bacillus subtilis (MNB2), Bacillus mojavensis ii (TB1) and Ochrobactrum sp. (EB1) which showed per cent inhibition of 91.2, 90, 89.4, 88 and 85 respectively. Furthermore, when these isolates were tested for growth promotion on tomato, the vigour index of tomato seedlings varied from 2110 (Trichoderma sp., PF2) to 1676 (Bacillus mojavensis, TB1). As next step, selected antagonists were tested for induction of systemic resistance (ISR) on tomato by conducting a pot culture experiment. Increased accumulation of peroxidase (PO), polyphenol oxidase (PPO) and, phenylalanine ammonia-lyase (PAL) was observed in plants treated with phylloplane antagonists. Higher activity of PO and PPO was induced by Trichoderma sp. (PF2) and B. subtilis (MNB2) at fifth day after inoculation (DAI) of pathogen. The phylloplane antagonists Trichoderma sp. (PF2) and B. subtilis (MNB2) recorded 169.54 min-1 g -1 and 152.56 min-1 g -1 respectively for PO and 199.19 min-1 g -1 and 172.24 min-1 g -1 respectively for PPO. Higher activity of PAL at fifth DAI was obtained by Pseudomonas fluorescens (KAU) followed by Trichoderma sp. (PF2) and B. subtilis (MNB2) recording 209.86 min-1 g -1 , 198.59 min-1 g -1 and 180.85 min-1 g -1 respectively. However, the highest per cent reduction of early blight after the first spray (68.58) was effected by Trichoderma sp. (PF2) followed by (64.92 and 60.22) B. subtilis (MNB2) and Ochrobactrum sp. (EB1), respectively. The disease reduction at early stage was reflected on yield of tomato and the same treatments; Trichoderma sp. (PF2), B. subtilis (MNB2) and Ochrobactrum sp. (EB1) gave 410 g, 389.33 g and 340 g fresh tomatoes per plot respectively. Hence these three phylloplane antagonists were selected for further evaluation. An experiment was carried out under rain shelter to evaluate the efficacy of the selected antagonists, conventional biocontrol agents such as Trichoderma sp. (KAU) and P. fluorescens (KAU) and a chemical check, propineb (0.2 %). The results showed that the phylloplane antagonists Trichoderma sp. (PF2) and B. subtilis (MNB2) offered significant effect against the disease recording 40.74 and 37.04 reduction in per cent disease severity (PDS) which in turn resulted in per cent yield increase of 32.63 and 28.10 respectively. Enumeration of phyllopane microflora proved that, there is drastic reduction in microbial population on leaf surface after iii spraying with propineb whereas the population increased after application of bioagents. Further, it was found that Trichoderma sp. (PF2) and B. subtilis (MNB2) are compatible with P. fluorescens (KAU) but B. subtilis (MNB2) is not compatible with Trichoderma sp. (KAU). Trichoderma sp. (PF2) is weakly inhibited by propineb (0.2 %), but azoxystrobin (0.1 %) could reduce its growth by 58 per cent. However, B. subtilis (MNB2) could not be affected by any of the two fungicides used against leaf blight. Hence, based on the results of various experiments in this study, phylloplane antagonists, especially, B. subtilis (MNB2) seems to be suitable for inclusion in IDM for early blight of tomato. Enumeration of culturable microflora on tomato leaves revealed that, bacteria are more on healthy leaves while fungi are more on the infected leaves. However, metagenomic analysis of the phylloplane revealed great difference in density and diversity of microbial taxa between healthy and infected leaves. This indicates that, specific functions of the phylloplane microbes and their antagonistic potential are also important in determining their efficacy as biocontrol agents. The study also revealed the presence of plant pathogenic fungal genera like Cladosporium, Corynespora, Pseudocercospora, Conidiosporomyces and Ustilago on tomato leaves. Both healthy and diseased tomato leaves harbour great microbial diversity and the phylloplane dwellers include well-known antagonists like species of Bacillus, Pseudomonas, Trichoderma and Penicillium. Presence of members of genera like Klebsiella and Enterobacter which are human pathogens was also revealed by metagenomic analysis. However, they are beneficial to plants as N fixers, P and K solubilizers and siderophore and IAA producers. Phylloplane dwellers also include species of Methylobacterium, Sphingomonas and Massilia and they are reported to produce different pigments and antibiotics besides being plant growth promoters, and antagonists to plant pathogens. Furthermore, species of Staphylococcus, Cladosporium, Coprinellus and Moesziomyces are also detected from tomato phylloplane and are reported to have antifungal properties with goo plant growth attributes, while species of Meira are reported as potential biological control agents against phytophagous mitesItem Management of banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica(Department of Plant Pathology, College of Agriculture ,Vellayani, 2024-03-20) Sinijadas, K.; Joy, MThe research work entitled “Management of Banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica” was carried out at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2018-2022. The objective was to evaluate the beneficial fungal root endophyte, P. indica for the management of Banana bract mosaic virus (BBrMV); and to elucidate the role of antioxidants and plastids in this tripartite interaction. The characteristic symptoms of BBrMV viz., reddish spindle shaped streaks on pseudostem, black necrotic streaks on peduncle, chlorotic spindle lesions on leaves and underdeveloped fruits were observed during the survey conducted in five agro-climatic zones (ACZ) of Kerala. Percent disease incidence (PDI) and vulnerability index (VI) of the disease recorded from different zones showed the highest in banana var. Nendran (PDI - 61.66 & VI – 44.03) from southern zone and lowest in var. Poovan (PDI – 7.29 & VI – 6.29) in central zone. Serological and molecular detection confirmed the presence of BBrMV in banana var. Nendran. The sequence similarity analysis of the coat protein (CP) gene of BBrMV southern zone isolate (Vellayani) showed 99.12 percent genetic closeness to its Tamil Nadu isolates compared to the Kerala isolates (98.38 percent). Screening of the most virulent strain of BBrMV from five different ACZ of Kerala was done based on early symptom development in TC banana plants var. Nendran. The BBrMV strain from southern zone could produce the symptoms on var. Nendran within 30 days (lowest) followed by central zone (55 days) on artificial inoculation with viruliferous aphids, Pentalonia nigronervosa. The cross-infection study of virulent strain of BBrMV (southern zone) of Nendran on Nendran developed chlorotic spindle lesions on leaves at 30 days after inoculation followed by Nendran on Robusta (75 days). P. indica-colonization in banana var. Nendran was carried out using standardized medium. The chlamydospores of the fungus were observed in roots at 20 days after colonization (DAC). In both in vitro and in vivo experiments, P. indicacolonized plants showed reduced disease severity irrespective of the virusinoculation stage with a vulnerability index of 6.7 percent in BBrMV (+Pi / +V); and 20.0 percent in the virus-infected plants post-colonized with P. indica (+V / +Pi) compared to 53.3 percent in the virus alone infected plants. Further, PCR analysis with BBrMV coat protein specific primer yielded amplicon of low intensity in P. indica-colonized plants inoculated with the virus compared to the control plants indicating the ability of the fungus to inhibit the virus. Further, P. indica precolonized plants inoculated with BBrMV had improved growth and yield parameters compared to non-colonized plants. Field trial was laid out with two treatments (P. indica-colonized and noncolonized banana plants var. Nendran) at Instructional Farm, College of Agriculture, Vellayani. P. indica-colonized plants recorded a drastic reduction in the severity of BBrMV by 33 to 58 percent compared to non-colonised control plants. Enhanced plant height (30 percent), collar girth (45 percent), number of leaves (25 percent), leaf length (30 percent), leaf width (27 percent), fresh weight of shoot (34 percent), number of secondary roots (62 percent), number of tertiary roots (76 percent) and root weight (86 percent) were observed in P. indica-colonized plants at 90 days after treatment. P. indica-colonization also improved the bunch weight (32.9 percent) and fruit quality. Biochemical detection of superoxides using nitroblue tetrazolium (NBT) and H2O2 with diamino benzidine (DAB) stains at 5, 10, 15, 30 and 45 days revealed a reduction in reactive oxygen species (ROS) accumulation in both P. indica-colonized plants challenged with BBrMV (+Pi / +V) and virus infected plants post-colonized with P. indica (+V / +Pi) compared to BBrMV alone. The decrease in ROS production and disease severity in the endophyte-colonized plants inoculated with the virus were attributed to the increased activities of antioxidant enzymes viz., peroxidase, superoxide dismutase, catalase, glutamate synthase and ascorbic acid oxidase. The molecular analysis of genes involved in the symptom development indicated the beneficial effect of P. indica on BBrMV infection in banana. P. indica reduced the symptoms by up-regulating chlorophyll biosynthesis gene (chlorophyll synthase-CHLG) and down-regulating chlorophyll degradation genes (chlorphyllase CLH1 & CLH2; and pheophytin pheophorbide hydrolase - PPH), carotenoidbiosynthesis genes (phytoene synthase-PSY1 and PSY2), carotenoid degradation gene (Phytoene desaturase - PDS) and the virus specific genes responsible for symptom development (Hc-Pro and P3). Thus, the present study reveals that P. indica enhances tolerance against BBrMV in addition to improved growth promotion, yield and fruit quality in banana plant.Item Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot diseases(Department of Plant Pathology, College of Agriculture,Vellayani, 2024-04-17) Josiya Joy; KAU; Radhika, N SThe research work entitled “Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot disease” was undertaken in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2019-24, with the objectives; molecular diagnosis and recombinant coat protein production of Papaya ringspot virus (PRSV), and evaluation of the efficacy of beneficial microorganisms and botanical inthe management of papaya ringspot disease (PRSD). Roving survey was carried out across five Agro-ecological units (AEUs) of Kerala. The disease incidence (DI) ranged from 50.25 per cent (Kayyur-Cheemeni) to 100 per cent (Kalliyoor, Venganoor, Balaramapuram, Pallichal, Kayamkulam, Mavelikkara, Velukkara, Irinjalakuda and Shoranur). Vulnerability index (VI) of the plants to PRSV in the surveyed locations ranged from 33.54 (Badiyadkka) to 98.22 (Kalliyoor). Serological and molecular detection confirmed the presence of PRSV in all the 20 symptomatic samples collected during survey. Phylogenetic tree constructed with the deduced amino acid sequences of CP gene of 11 Kerala PRSV isolates, revealed that Thiruvananthapuram isolates clustered together, indicating their relatedness during evolution. Mechanical inoculation on two months old Red Lady papaya seedlings under insect proof conditions was carried out to identify the most virulent PRSV isolate collected from different AEUs. At three months after inoculation (MAI), Kalliyoor isolate exhibited highest VI (96.63) followed by Alur (95.48) and Venganoor (95.22) isolates. The lowest VI was observed in Kayyur-Cheemeni isolate with 57.95 VI, followed by Cherpulassery (60.58). The recombinant coat protein of PRSV was induced in pLATE 31 expression vector with C terminal histidine tag, within BL21(DE3)pLysS expression host. PRSV recombinant coat protein was purified using Ni-NTA column chromatography. A single band was observed at 35 kDa in SDS-PAGE analysis and western blotting with PRSV antiserum, confirmed the presence of purified recombinant coat protein in the soluble fraction. Pot culture experiment was conducted to evaluate the management of PRSD using Piriformospora indica. The initial colonization of P. indica inside the papaya roots was observed five days after germination of the seeds grown in P. indica massmultiplied medium. Prophylactic colonization of P. indica exhibited lowest VI (23.10) and 68.27 per cent reduction in VI over control diseased plants at five months after PRSV inoculation (MAI). Double antibody sandwich - Enzyme linked immunosorbent assay (DAS-ELISA) at 5 MAI revealed lowest absorbance (0.23) indicating lowest virus titre in P. indica pre-colonized plants upon PRSV inoculation, compared to control diseased plants (1.23). The accumulation of reactive oxygen species (ROS) i.e., H2O2 and superoxides in the leaves were analyzed using DAB (diaminobenzidine) and NBT (nitro blue tetrazolium chloride) staining respectively. P. indica-colonized plants upon PRSV inoculation indicated a higher initial accumulation of ROS at three weeks after inoculation (3 WAI) and further reduction at 6, 9 and 12 WAI of PRSV, compared to control diseased plants. P. indica-colonized plants also exhibited enhanced antioxidant defence enzyme activity viz., catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, glutamate synthase and superoxide dismutase compared to control diseased plants. Amongst PRSV inoculated treatments, P. indica pre-colonized plants exhibited highest number of leaves (20.86), leaf area (365.14 cm2), plant height (108.29 cm), stem girth (7.26 cm), shoot biomass (423.43 g), root biomass (133.96 g) and chlorophyll content (2.84 mg g-1 of fw) at five months after transplanting (5 MAT). Effect of P. indica, Bougainvillea spectabilis leaf extract (10 %) and Pseudomonas fluorescens (2 %) were evaluated against natural incidence of PRSD under field conditions at Instructional farm, College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram, Thiruvananthapuram. P. indica-colonized plants exhibited lowest VI (37.63), with highest reduction in VI over control (53.85 %) followed by B. spectabilis treated plants (37.49 %) and P. fluorescens treated plants (33.31 %) at 12 months after planting (12 MAP). In DAS-ELISA, lowest virus titre with absorbance of 0.438 was observed in P. indica-colonized plants at 12 MAP, compared to the highest virus titre in control plants (1.267). B. spectabilis treated plants (0.596) also exhibited reduction in virus titre followed by P. fluorescens treated plants (0.625) at 12 MAP. P. indica-colonized plants exhibited enhancement in growth parameters viz., number of leaves (24.00), leaf area (1127 cm2), stem girth (42.74 cm) and plant height (207.39 cm) at 12 MAP. P. indica-colonized plants also enhanced the yield by 44.68 per cent followed by B. spectabilis treated plants (24.13 %) and P. fluorescens treated plants (17.99 %). Moreover, fruits from P. indica-colonized plants expressed significantly superior quality parameters. Thus, findings from the present study could aid in the preliminary detection and management of the virus, thus mitigating the widespread infection caused by PRSV. The recombinant PRSV coat protein produced in this study could be used for the development of PRSV antiserum. Additionally, our research highlights the efficacy of eco-friendly management strategies for papaya ringspot disease, with P. indica-colonization @ 106 cfu g-1 and also with four foliar sprayings as well as soil drenching of B. spectabilis leaf extract (10 %) applied at fortnightly intervals, starting from one month after planting. More field trials are to be conducted to integrate this strategy in integrated disease management (IDM) package for the effective and sustainable management of PRSD in papaya.Item Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by colletotrichum capsici (Syd.) butler and bisby(Department of Plant Pathology, College of Agriculture, Vellayani, 2024-07-05) Elizabeth, T Jojy; KAUThe research work entitled “Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by Colletotrichum capsici (Syd.) Butler and Bisby” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2017- 2023. The study was undertaken with the objective to evaluate P. indica- and its water diffusible exudates- primed chilli seedlings and plants against foliar infection of C. capsici; and study the biochemical and molecular mechanisms involved in this tripartite interaction. A survey conducted in the five agroclimatic zones (ACZs) of Kerala showed that the highest disease incidence (DI) and Percent Disease Index (PDI) were recorded at RARS, Pilicode (DI-90 & PDI-52.60) of northern zone, while the lowest DI was observed at farmer’s field, Kottarakkara (20%) and ORARS, Kayamkulam recorded the lowest PDI (23.63). Chilli anthracnose symptoms namely leaf spot, leaf blight, die-back, fruit rot and mummified fruits were observed in different survey locations of the five agroclimatic zones (ACZ) of Kerala. Nine C. capsici isolates and one isolate of C. gloeosporioides were obtained from the surveyed locations. All the C. capsici isolates produced sparse mycelial growth with concentric zonations of acervuli on potato dextrose agar (PDA) medium. The upper side of culture plates appeared in different shades of white and off-white to grey with regular or irregular margins, while the reverse side looked yellowish brown to black. Isolate Cc4 produced maximum mycelial growth diameter of 8.6 cm compared to 7.2 cm (minimum) in Cc2 on 7th day after inoculation (DAI). Further, microscopic characters such as mycelial width, size of conidia, acervuli, appressoria, number and length of setae were significantly different in the C. capsici isolates. The most virulent isolate of C. capsici was screened based on the lesion size produced on the artificially inoculated leaves and fruits of chilli var. Vellayani Athulya. On 7th DAI, Cc3 isolate of C. capsici produced maximum lesion size of 2.52 cm while isolate Cc4 produced minimum lesions (0.74 cm) on 292 inoculated chilli leaves. Similarly, tender, mature and ripe fruits of var. Vellayani Athulya inoculated with Cc3 isolate produced the highest lesion size (2.48, 2.34 and 2.56 cm respectively) and isolate Cc4 recorded the lowest lesions (1.62, 1.16 and 1.38 cm respectively) on 7th DAI. Thus, isolate Cc3 from Thrissur, was selected as the most virulent isolate of C. capsici. Ten selected KAU/ICAR released varieties of chilli were screened with Cc3 isolate by detached leaf and fruit inoculation method to identify the most susceptible variety. Leaves of Vellayani Athulya recorded the highest lesion size of 2.80 cm on 5 DAI compared to 1.66 cm (lowest) in Manjari. Maximum lesion size of 2.96 cm was observed on the tender fruits of Vellayani Athulya and minimum was recorded on Vellayani Thejus (1.52 cm). Lesions of 3.50 cm (highest) were observed on mature Vellayani Athulya fruits as against 2.00 cm on Manjari fruits. Similarly, ripe fruits of Vellayani Athulya recorded maximum lesion size of 3.46 cm and minimum in Vellayani Samrudhi (1.30 cm) at 7 DAI. Also, in the in vivo study, higher lesion size (3.00 cm) was observed in Vellayani Athulya fruits and lowest (1.62 cm) in fruits of Vellayani Samrudhi at 7th DAI. Standardization of P. indica-colonization in chilli var. Vellayani Athulya was done on plant nutrient medium (PNM) and chilli roots were sampled at different intervals viz., 3rd, 5th, 7th, 10th, and 15th days after colonization (DAC). Young, double-walled chlamydospores were observed within the root cells at 5 DAC. P. indica colonization gradually increased and reached a maximum of 100 per cent at 15 DAC. Dual culture of P. indica and C. capsici showed the appearance of inhibition zone (8.33 DAI) and antibiosis (12.67 DAI) at the point of interaction of the two fungi. Further microscopic observations revealed thickening and lysis of pathogen mycelium by P. indica. The growth of C. capsici was suppressed (3.85 cm) in dual culture compared to normal (6.28 cm) in control at 7th DAI. 57.22 per cent mycelial growth inhibition due to P. indica was observed at 15 DAI. Antagonistic property of P. indica-water diffusible exudates (Pi-WDE) against C. capsici was studied by in vitro poison food technique. Significant 293 differences were observed in the colour and nature of C. capsici growth on PDA media, added with different day-old Pi-WDE in comparison with control. Among them, ten day-old Pi-WDE recorded the highest inhibition (67.30 %) of pathogen mycelial growth whereas the lowest in one day-old Pi-WDE (9.29 %). Mass multiplication of P. indica in the portray medium was carried out. Rapid growth of the endophyte was recorded in a combination of farmyard manure (FYM) and coir pith (1:1), added with 2 per cent gram flour. P. indica completely covered the inoculated trays within 7 DAI. This medium was used to colonize chilli plants with P. indica for further experiments and thus, four treatments viz., P. indica alone (T1), C. capsici alone (T2), P. indica-primed seedlings + C. capsici (T3) and control (T4) were systematized for the in vitro and in vivo evaluation experiments. In vitro study of P. indica-primed chilli seedlings against C. capsici significantly reduced anthracnose lesion size and disease severity to 1.30 cm and 30.63 per cent respectively compared to 2.06 cm and 77.00 per cent in C. capsici alone at 7 DAI. Maximum root and shoot weight were recorded in the P. indicacolonized plants, irrespective of the pathogen. Pot culture experiment was also carried out in the chilli var. Vellayani Athulya. The development of anthracnose symptoms on leaves was delayed by 4 days in P. indica-colonized chilli plants as against 2 days in control. The lesion size and disease severity due to anthracnose was lower in P. indica-colonized chilli leaves (1.58 cm & 54.90 %) compared to C. capsici alone (2.86 cm & 85.20 %) at 10 DAI. Similarly, minimum lesion size and disease severity of 1.17 cm and 27.92 per cent was observed in fruits from pathogen inoculated P. indicacolonized chilli plants than in C. capsici alone (3.56 cm & 91.67 %) at 10 DAI. Field studies were conducted in rabi and summer seasons with two treatments viz., P. indica-colonized and non-colonized plants. P. indica-colonized chilli plants recorded lower anthracnose severity of 27.00 per cent in Rabi and 15.50 per cent in summer compared to 56.50 and 47.00 per cent respectively in control. P. indica colonization improved the biometric characters such as plant height, number of leaves, leaf area, number of secondary and tertiary roots, shoot 294 weight, root weight in primed chilli plants. In addition, yield of chilli was enhanced in endophyte-colonized plants compared to control. Biochemical studies on the mechanism of disease management in P. indica-colonized chilli plants at 0, 12, 24 and 72 hours after inoculation (HAI) showed increased activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). In contrast, the activities of 4-coumaryl CoA ligase (4-CL) and polyphenol oxidase (PPO) were enhanced in chilli plants inoculated with C. capsici alone. Isoenzyme analysis revealed higher induction of isoenzymes of PO and PPO in the P. indica-colonized plants compared to control. Enhanced induction of pathogenesis related (PR) proteins was noticed in the P. indica-colonized plants inoculated with the pathogen at 72 HAI. The capsaicin content was drastically reduced in C. capsici inoculated chilli fruits. However, fruits from P. indica-colonized plants had high capsaicin content (>30% over control) irrespective of infection. Molecular studies of the defense related genes revealed the positive role of P. indica in the management of anthracnose disease in chilli. P. indica reduced anthracnose symptoms in colonized chilli plants by the upregulation of genes involved in phenyl propanoid pathway (PAL1, 4-CL, CAD and CHS), jasmonic acid signaling pathway (PDF1.2 and AOS) and salicylic acid signaling pathway (PR1, EDS1 and PAL3); and downregulating the LOX gene involved in jasmonic acid signaling. In summary, P. indica and its WDE inhibited C. capsici under in vitro conditions. Anthracnose incidence and severity were considerably reduced in the treated chilli seedlings and plants compared to control. In addition, P. indica enhanced growth as well as yield in colonized chilli plants, thereby enhancing the fruit quality. Further, the endophyte improved disease resistance in chilli plants by increasing the defense-related enzyme activities and expression of defense genes. Thus, the root fungal endophyte, P. indica can be considered as an efficient biocontrol agent in the management of chilli anthracnose.Item Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; Vimi LouisThe present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.Item Characterization and integrated management of fusarium oxysporum f.sp. cubense (E.F. Smith) synder and hansen causing fusarium wilt disease of banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Lishma, N P; Anita Cherian, KItem Management of early blight disease of tomato (Solanum lycopersicum L.) under protected cultivation(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Sumbula, V; Sainamole Kurian, PItem Deterioration of oil cake by fungi(Department of Plant Pathology, College of Agriculture, Vellayani, 1989) Naseema, A; Wilson, K IItem Study of bacterial leaf spot of betel vine- biochemical changes and control(Department of Plant Pathology, College of Agriculture, Vellayani, 1986) Koshi Abraham; James MathewThe bacterial leaf spot is one of the most serious diseases of betel vine in Kerala. The bacterium is one of the most serious disease of betal vine. Confidering the seriouness of the disease , studies were undertaken on the different aspects of the disease and to find out a suitable control /management practice.